Ladakamycin and process for preparing same

ABSTRACT

NEW ANTIBIOTIC LADAKAMYCIN AND A MICROBIOLOGICAL PROCESS FOR THE PRODUCTION THEREOF USING THE MICROORGANISM STREPTOVERTICILLIUM LADAKANUS BAR. LADAKANUS. LADAKAMYCIN CAN BE USED TO INHIBIT THE GROWTH OF VARIOUS BACTERIA, FOR EXAMPLE, PSEUDEOMONAS AERUGINOSA, PROTEUS VYLGARIS, AEROBACTER AEROGEMES, AND ESCHERICHIA COLI.

June 11, 1974 I BERGY ET AL 3,816,619

LADAKAMYCIN AND PROCESS FOR PREPARING SAME Filed Aug. 30, 1965 FIGURE 1.INFRARED ABSORPTION SPECTRUM LADAKAMYCIN FREQUENCY CM- I 1 1 1 I 1 1 s a2 8 .3 8 a a e NOISSIWSNVELL MALCOLM E. BERGY LADISLAV J. HANKA ROSS R.HERR DONALD J MASON INVENTORS ATTORNEYS lymphoma.

United States Patent C) 3,816,619 LADAKAMYCIN ANDPROCESS FOR PREPARINGSAME Malcolm E. Bergy, Ladislav J. Hanka, and Ross R. Herr, Kalamazoo,and Donald .1. Mason, Portage, Mich, assignors to The Upjohn Company,Kalamazoo, Mich. Continuation-impart of abandoned application Ser. No.

482,472, Aug.'25',1965. This application Aug. 30, 1965, Ser. No.483,723.

' Int. Cl. A61k 21 4 C07d 51/52 US. Cl. 424-116 Claims ABSTRACT OF THEDISCLOSURE New antibiotic ladakamycin and a microbiological process forthe production thereof using the microorganism Streptoverticilliumladakanws var. ladakanus. Ladakamycin can'be used to inhibit the growthof various bacteria, for example, Pseudomonas aerugz'nosa, Proteusvulgaris, Aerobacter aerogenes, and Escherichia coli.

Ladakamycin is an organic compound producible by culturing aladakamycin-producing actinomycete in an aqueous nutrient medium. his abasic substance which has the property of adversely affecting the growthof certain organisms, particularly bacteria, for example,

Pseudomonas aeruginosa, Proteus vulgaris, Aerobacter aerogenes,Salmonella gallinarum, Salmonella pullorum, Salmonella schvttmuelleri,and Escherichia coli, and can be used alone or in combination with otherantibacterial agents to prevent the growth of, or reduce the number of,such organisms present in various environments. For example, it can beused in papermill systems to inhibit the growth of Aerobacter aerogeneswhich is known to produce slime in such systems. It is also useful as anoil preservative, for example, as a bacteriostatic agent for inhibitingthe growth of Proteus vulgaris which is known to cause spoilage incutting oils. Also, it is useful in wash solutions for sanitationpurposes, as in the washing of hands and the cleaning of equipment,floors, or furnishings of contaminated rooms or laboratories; it is alsouseful as an industrial preservative, for example, as a bacteriostaticrinse for laundered clothes and for impregnating paper and fabrics; andit is useful for suppressing the growth of sensitive organisms in plateassays and other microbiological media. It can also be used as a feedsupplement to promote the growth of animals.

Ladakamycin is also active in mice against T-4 THE MICROORGANISM Theactinomycete used according to this invention for the production ofladakamycin has been designated as Streptoverticillium ladakanus var.ladakanus sp. nova. One of its strain characteristics is the productionof ladakamycin. A subculture of the living organism can be obtained fromthe permanent collection of the Northern Utilization and ResearchDivision, Agricultural Research Service, The US. Department ofAgriculture, Peoria, Ill., U.S.A. Its accession number in thisrepository is NRRL 3191. The characteristics of Streptoverticilliumladakan us var. Zadakanus sp. nova., NRRL 3191, are given in thefollowing tables:

3,816,619 Patented June 11, 1974 ice Table I--Appearance on EktachromeTable II-Assimilation of Carbon Compounds in Synthetic Medium (I. Bact.,56: 107- 114, 1948).

Table III-Cultural Characteristics Table IV-ColorCharacteristics-According to the Color Harmony Manual, 3rd Edition,1948, and the ISCC- NBS Method of Designating Colors and a Dictionary ofColor Names, NBS Circular 553 Table V-Micr0sc0pic Characteristics TABLEI.--APPEARANCE ON EKTACHRO'ME S. ladakanus v. ladakanus Agar medium:Appearance Bennetts Cott0ny white aerial growth.

Yellow reverse.

Czapeks sucrose Trace cottony white aerial growth. Colorless reverse.

Maltose tryptone C otto ny white aerial growth. Yellow reverse.

Peptone iron No aerial growth. Tan reverse.

0.1% tyrosine C ottony white aerial growth. Colorless reverse.

Casein starch C o tto my white aerial growth. Colorless reverse.

Deity, A., "Ektachrome Transparencies as Aids'in ActinomyceteClassification, Annals of the 1V.Y. Academy of Sciences, 60: 152-154,1954. p v I I A TABLE II [Assimilation of carbon compounds in syntheticmedium by S. ladaka'nus v. ladakanus Run number 1 2 3 Control 1.D-xylose (g 2. E-arabinose... 3. rharnnose 4. l -fructose 6.Q-galaetose... 6. B-glucose 7. l -mannose 8. ma1tose-.-..- 1 13 flmrtrin14 insulin 15. soluble starch 16. glycerol 17 rlnloifnl 18. -mannitol19. g-sorbitol 20. inositol 21. saliein 22 phennl 23. eresol 24. Natormate 25. Na oxalate..-

26. Na tartrate"--.

27. Na salicylate 28. Na acetate 29. Na citrate 0. Na succinate TABLEIII [Cultural Characteristics of S. ladakanus v. ladakanus] Agar mediumRun No. 1 Run No. 2 Run No. 3 Run No. 4

Peptone iron. No aerial growth; Colorless No aerial growth; Colorless Noaerial growth; Colorless No aerial growth; Pale tan vegetative fleckedwith red; vegetative with traces of vegetative growth; Melaninvegetative; Melanin =i=. Melanin negative. red; Melanin negative.negative.

Calcium malate Trace white aerial growth; Pale gray-white aerial Tracewhite aerial growth;

White reverse; Malate not growth; Pale gray-white White reverse; Nopigsolubilized. reverse; Malate not solument; Malate not solubilizebilized.

Glucose asparagine.-.... Gray-white aerial growth; Pale gray-pink aerialrowth; Trace cream-white aerial Pale cream reverse. Yellow reverse; Yeow growth; Yellow reverse;

gment. Yellow pigment.

Skim milk Trace white aerial growth; Pale gray-white aerial Very slighttrace white aerial Very slight trace white aerial Colorless vegetativegrowth; Yellow reverse; growth; Yellow reverse; growth; Yellow reverse;growth; Yellow pigment; Casein not solubilized. Yellow pigment; CaseinYellow pigment; Casein Casein not solubilized. not solubilized. notsolubilized.

Nutrient starch Cottony cream-white aerial Cottony white aerial growth;

growth; Yellow reverse; Yellow reverse; Yellow pig- Yellow pigment.ment; Starch hydrolyzed.

Maltese tryptone- Cottony cream-white aerial Cottony cream-white aerialYellow vegetative growth;

growth; Yellow reverse; growth; Yellow reverse; Yellow pigment. Yellowpigment. No pigment.

Bennett's Cottony cream-white aerial Cottony cream-white aerial Cottonywhite aerial growth;

gowth; Yellow reverse; growth; Deep yellow re- Yellow vegetative; N o

ellow pigment. verse; Yellow pigment. pigment.

Czapek's sucrose Cottony pale-gray aerial Cottony white aerial growth;growth; Pale gray reverse. White reverse; No pigment.

Xanthine Trace white aerial growth; Trace white aerial growth; Paleyellow vegetative Cottony white aerial growth;

Yellow reverse; Yellow Pale tan reverse; Pale tan growth; Pale yellowPale yellow vegetative pigment; Xanthine not pigment; Xanthine notpigment; Xanthine not growth; Pale yellow pigsolubilized. solubilized.solubilized. ment; Xanthine not solubilized.

Tyrosine Cottony white aerial owth; Gray-pink aerial growth; Pale yellowvegetative Yellow reverse; Yel ow pig- Olive reverse; Trace yellowgrowth; Pale yellow pigment; Tyrosine solubilized. giigimrnt; Tyrosinesolumgnt;1 Tyrosine not solu- 1 ze ze Litmus milk Gray-pink aerialgrowth; Slight trace gray aerial Lavender reverse; Lavender growth; Redlavender re pigment; Casein solubilized. verse; Red lavender pigment;Casein solubilized.

Purple m Gray-cream aerial growth; Blue-green reverse; Blue- Olivereverse; Blue-green green pigment; Casein not pigment; Caseinsolubilized. solubilized.

Casein star h Cream-white aerial growth; Cottony pink-white aerialPink-tan reverse; N o pigment; Starch hydrolyzed.

Tomato Paste tmeal Color Harmony Manual, 3rd Ed.

ISCC-NBS Color Names growth; Pale yellow reverse; No pigment.

Cottony white aerial growth Yellow reverse; Yellow pigment.

solids, soybean meal, cottonseed meal, corn meal, milk solids,pancreatic digest of casein, distillers solubles, fish meal, animalpeptone liquors, meat and bone scraps, and the like. A combination ofthese carbon and nitrogen sources can be used advantageously. Tracemetals, for example, zinc, magnesium, manganese, cobalt, iron, and thelike need not be added to the fermentation since tap water andunpurified ingredients are used as media com- Production of the compoundof the invention can be eifccted at any temperature conducive tosatisfactory growth of the microorganism, for example, between aboutBennett's agar:

Surface.--" Gray b-oyster white.-- 263m white; 264g hght gray. Reverselba-yellow tint 92m yellowish white; l21gm pale yellow-green.2fb-bamboo, bufl, 87g moderate yellow; 89m

straw, wheat. pale yellow. ponents. Czapek's su crose agar:

Surface--." Gray b-oyster white--- 263m white; 264g light gray. Reverse.-do 263m white; 264g light gray. Maltose-tryptone agar:

Surtace Gray b-oyster white--- 263m white; 264g light gray. Beverse-Ilia-yellow tint- 92m yellowish white; 121gm pale yellow-green.2fb-bamboo, bufl, 87g moderate yellow; 89m

straw, wheat. pale yellow.

TABLE V Microscopic characteristics of S. ladakanus v. ladakanus Lightmicroscope Sporophores bivcrticillatc. Electron microscope:

Direct Long, smooth spores. Carbon replica Long, smooth spores withsurface ridging.

The new compound of the invention is produced when the elaboratingorganism is grown in an aqueous nutrient medium under submerged aerobicconditions. It is to be understood also that for the preparation oflimited amounts surface cultures in bottles can be employed. Theorganism is grown in a nutrient medium containing a carbon source, forexample, an assimilable carbohydrate, and a nitrogen source, forexample, an assimilable nitrogen compound or proteinaceous material.Preferred carbon sources include glucose, brown sugar, sucrose,glycerol, starch, corn starch, galactosc, dextrin, molasses, and thelike. Preferred nitrogen sources include com steep liquor, yeast,autolyzed brewers yeast with milk 18 and 40 C. and preferably betweenabout/25 and 30 C. Ordinarily, optimum production of the compound isobtained in about 2 to 10 days. The medium normally stays fairly closeto neutral, or on the acid side during the fermentation. The final pH isdependent, in part, on the buffers present, if any, and in part on theinitial pH of the culture medium which is advantageously adjusted toabout pH 6-8 prior to sterilization.

When growth is carried out in large vessels and tanks, it is preferableto use the vegetative form, rather than the spore form, of themicroorganisms for inoculation to avoid a pronounced lag in theproduction of the new compound and theattendant inefficient utilizationof equipment. Accordingly, it is desirable 'to produce a vegetativeinoculum in a nutrient broth culture by inoculating the broth culturewith an aliquot from a soil or slant culture. When a young, active,vegetative inoculum has thus been secured, it is transferred asepticallyto large vessels or tanks. The medium in which the-vegetative inoculumis produced can be the same as, or different from, that utilized for theproduction of the new compound, as long as it is such that a good growthof the microorganism is obtained.

The new compound of the invention is a basic compound having theempirical formula C H N O At room temperature, ladakamycin is soluble tothe extent of ap proximately 40 mg./ml. in water but is soluble at lessthan 1 mg./ml. in methanol and higher alcohols, acetone, chloroform,hexane, and dimethylsulfoxide.

A variety of adsorbents can be used in the isolation and purification ofladakamycin. In a preferred process, ladakamycin is recovered from itsculture medium by separation of the mycelia and undissolved solids byconventional means such as by filtration or centrifugation. Theantibiotic is then removed from the filtered or centrifuged broth by theuse of surface active adsorbents, for example, decolorizing carbon ordecolorizing resins, and elution of the adsorbed material with asolvent. Any of the solvents mentioned above in which ladakamycin issoluble can be used. A suitable decolorizing resin is Permutit DR (U.S.Pat. 2,702,263). The eluates obtained from the surface active adsorbentscan be evaporated to dryness to provide an impure preparation of theantibiotic ladakamycin. This preparation can be used in environmentswhere higher purity of the antibiotic is not necessary.

High purity ladakamycin can be obtained by subjecting an impurepreparation of ladakamycin, as obtained above, to partitionchromatography using the following solvent system: n-butanol (7.5);ethyl acetate (2.5); pH 6.0 McIlvaine buffer (3.5). The fractionsobtained from partition chromatography are freeze-dried and then can besubjected to silica gel chromatography using solvents such as chloroformand methanol to efiect a higher degree of purification. Fractionsobtained from silica gel chromatography can be concentrated to obtaincrystalline ladakamycin.

The new compound of the invention, ladakamycin, is active againstEscherichia coli and can be used to reduce, arrest and eradicate slimeproduction in papermill systems caused by its antibacterial actionagainst this microorganism. It can also be used to prolong the life ofcultures of Trichomonas foetus, Trichomonas hamz'nis, and T richomonasvaginalis by freeing them of Escherichia coli contamination. Driedfermentation whole beers containing ladakamycin can be used in chickenfeed at the rate of 100 mg. per pound of feed to improve the weightgains in chickens and thus promote improved feed utilization. Arepresentative feed experiment showed a weight gain of 4.2% in chickensand improvedfeed utilization of 1.8% when dried ladakamycin fermentationwhole broth was incorporated in feed at 100 mg. per pound of feed.

The following examples are illustrative of the process and products ofthe present invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions by volumeunless otherwise noted.

EXAMPLE 1 (A) Fermentation G./ liter Glucose monohydrate Yeastolac 1 10N-Z Amine B 2 5 Tap water, q.s., 1 liter.

1 A protein hydrolysate of yeast cells. 2 A bulk peptone in powder formobtained by the pancreatic digestion of casein. g

The pre-seed medium pre-sterilization pH was 7.2. The pre-seed inoculumwas grown for three days at 28 C. on a Gump rotary shaker operating at260 r.p.m.

The pre-seed inoculum ml.) was used to inoculate a 40-liter seed tankcontaining 20 liters of the following sterile seed medium:

Pharmamedia is an industrial grade of cottonseed flour produced byTrader's Oil Mill Company, Fort Worth, Tex.

Wilsons Peptone Liquor No. 159 is a preparation of enzymaticallyhydrolyzed proteins of animal origin.

The pro-sterilization pH of the seed tank medium was 7.2. The seedinoculum was grown for 24 hours at a temperature of 28 C. with aerationat the rate of 10 standard liters per minute and stirring at the rate of400 r.p.m.

A portion of theseed inoculum (12.5 liters), described above, was thenused to inoculate a 400-liter fermentor containing 250 liters of thefollowing sterile fermentation medium:

The pre-sterilization pH of the fermentation tank medium was 7.2. Thefermentation cycle was 5 days during which time the temperature wascontrolled at 28" C., filtered air was supplied at a rate of 200standard liters per minute, and agitation was at the rate of 280 r.p.m.Sterile lard oil was added to control foaming.

(B) Recovery The whole broth from a ladakamycin fermentation, asdescribed above, was slurried with 3% of its weight of diatomaceousearth and filtered. The cake was washed with one-tenth volume of waterand the wash added to the filtered beer. Activated carbon (6% W./v.) wasadded to the combined filtered beer and wash. This slurry was stirredfor 30 minutes and then filtered with the aid of a filter aid asrequired. The cake was washed with onetenth volume of water and the washadded to the filtrate. The remaining cake was stirred with one-thirdoriginal beer volume of 50% aqueous acetone for 15 to 20 minutes andthen the slurry Was filtered. This operation was repeated twice toproduce three filtrates which were combined. The combined filtrates werereduced to onetwentieth volume under reduced pressure (less than 30 C.).The resulting concentrate was then poured into a stirring mixture of 5volumes of acetone with 5% diatomaceous earth. The solids were removedby filtration and the filtrate concentrated to an aqueous solution andfreeze-dried to obtain a crude preparation of ladakamycin. The recoveryprocess, described above, gave the iollowing balance sheet on aladakamycin fermentation roth:

Dry preparation of ladakamycin 1 575 mg. 2 2.25 biounits/mg.

- The ladakamycin content of the various preparations in the recoveryprocess described above was ascertained by a microbiological assay usingthe organism" Salmonella schot'tmuelleri. The assay procedure is asfollows:

' Nutrient agar was inoculated with an 1820 hour culture of S.schottmuelleri, grown in nutrient broth on the reciprocating shaker at37 C., at the rate of 0.2 m1. of the culture per'100 ml. agar. Theseeded medium was dispensedinto 100 K 20 mm; plastic petri dishes in8-rnl. portions and the agar was allowed to solidify. The fermentationbeers were tested at full strength and diluted /2, /4 and V8 in the 0.1M phosphatebuifer o'f'pH 6.0. Each solution was applied to one paperdisc (12.7 mm.) on each of two replicate plates and the plates wereincubated overnight at 30 C. The zones of inhibition were then measuredand the potency of each sample was expressed in biological units. Thebiological unit (BU) is defined as the concentration of the antibioticwhich gives a 20 mm. zone of inhibition under the standard assayconditions. Thus, if for instance a fermentation beer has to be diluted,4 to give the 20 mm. zone of inhibition, the potency of such beer is100 BU per ml.

(C) Purification 1) Partition chromatography.A crude preparation ofladakamycin, as obtained above, was passed over a partition columnprepared as follows: The solvent system consisted of n-butanol (750),ethyl acetate (250), and pH 6.0 McIlvaine bu'ifer (350). 150 g. ofdiatomaceous earth was slurried in the upper phase and the slurry Washomogenized with 60 ml. of lower phase. A two inch (I.D.) column waspacked with this preparation to a constant height of approximately 13inches using air pressure. g. of a dry crude preparation of ladakamycin,as obtained in Part B above, was dissolved in 10 ml. of lower phase.This was homogenized with 20 g. of diatomaceous earth and upper phase.The slurry was then carefully poured onto the top of the preparedpartition column bed and packed with air pressure. The column wasdeveloped with upper phase and 20 ml. fractions were collected. Theactive fractions, as determined by the microbiological assay against themicroorganism Salmonella schottmuelleri, as described above, were pooledand two volumes of Skellysolve B (isomeric hexanes)'was added. Theaqueous phase was separated and the upper phase was then rinsed withabout .05 volume of water. The aqueous phases and water washes werecombined and freeze-dried; yield, 1.30 grams of ladakamycin assaying12.7 biounits/mg. against S. schottmuelleri.

(2) Silica gel chromatography.--(a) Column preparation: The materials inthe silica gel column were as follows:

Buffer salts: KH PO and Na HPO Silica gel: Silica gel No. 7734 (E. MerckAGDarmstadt) 0.05-0.20 mm. for chromatography Solvents Chloroform,methanol.v

Each kilogram of silica was buffered by mixing thoroughly with 800 ml.of an aqueous solution containing 27.2 g. of KH PO and 28.4 g. of Na HPOThe water was removed by drying at less than 100 C. and then the gel wasactivated at a temperature'of 120-l30 C. for at least two hours. Afterthe buffered and activated silica gel was cooled, it was slurried inenoughchloroform to provide a pourable mixture, yet thin enough inconsistency to allow gas pockets to escape. This slurry was poured intoa column and packed to a constant height using air pres sure. A goodhead of chloroform was left on the top.

(b) Preparation of starting material: For each kilogram of silica gelused in the preparation of the column above, 7.3 g. of ladakamycinpreparation, as obtained in Part C(l) above, was dissolved in 40 ml. ofmethanol and mixed thoroughly with g. of buffered, activated silica gel.The methanol was removed by evaporation in air at room temperature, andthe dried silica gel containingthe crude ladakamycin was distributedevenly into the head of chloroform remaining on the column bed. Thelevel of chloroform was then drained to-approximately 6 inches above thelevel of the layer of starting material.

(c) Development of the column: The silica gel column was developed bycarefully layering the developing system consisting of chloroform andmethanol (7:3) onto thetop of the chloroform and starting the flow. Thecolumn was developed with this system at a flow rate of approximately666 ml./ hour/ kilogram column bed; 133 mL/kilogram column bed fractionswere collected during the develop.- ment. All the operations during thedevelopment stage were performed at room temperature. The collectedfractions and the crystalline ladakamycin which resulted therefrom werekept cold (less than -20 C.).

The fractions collected from the silica gel column were assayed byevaporating 1 ml. portions from each fraction to dryness andreconstituting the residue in waten'Those fractions assayingapproximately 6.0 biounits/ ml. and greater against the organism E. coliwere concentrated to about volume in vacuo at less than 40 C. Thisconcentrate was purified by filtration and the concentration wascontinued to approximately 1 volume. Crystallization of ladakamycinoccurred during this concentration. The ladakamycin crystals wereremoved by filtration, washed lightly with methanol, and dried in meat).at room temperature to a constant weight. Recrystallization of theladakamycin crystals was achieved by dissolving the ladakamycin crystalsin boiling methanol. at a rate of. .1 mg./ml. The solution was purifiedby filtration and concentrated in vacuo at less than 40 C. to 25'mg./m1.The concentrate was refrigerated overnight and ladakamycin crystalswhich precipitated were removed by filtration, washed lightly withmethanol, and then with chloroform. The ladakamycin crystals were driedin vacuo to a constant weight.

The E. coli microbiological assay disclosed above is as follows:

The microorganism was grown for 18-20 hours at 30 C. on a reciprocatingshaker in the following synthetic medium: K HPO 3.5 g.; KH PO 1.5 g.;MgSO -7H O, 0.1 g.; (NH SO 1.0 g.; glucose, 2.0 g.; distilled Water, 1liter.

The synthetic agar used for assay was prepared by further supplementingthis synthetic brothwith 15 g. of agar per liter. This agar wasinoculated at the rate of 0.3 ml. of the culture per 100 ml. of agar.The rest of the assay procedure is identical with that describedpreviously for S. schottmuelleri. The assay employing E. coli grown onsynthetic medium is about seven times more sensitive than the assay withS. schottmuelleri grown in nutrient agar.

The silica gel chromatography procedure, described above, was applied tocrude preparations of ladakamycin obtained from the partition column asdescribed ,in Part C( 1). However, this same procedure has'also beensucessfully applied to crude ladakamycin preparationsobtained from therecovery operation described in Part B above.

Chemical and physical properties of ladakamycin Calculated molecularweight: 244.2.

Ultraviolet spectrum: Ladakamycin has the following UV absorptionspectrum:

Water: max. at 241 mg, 11:35.9. .OlN HCl: max. at 249 m 11:12.6. .OlNKOH: max. at 223 mu, 12:99.1. sh: at 253 m 11:6.3.

Melting point: 228-230 C.

Solubility: Ladakamycin is soluble at room temperature to the extent ofapproximately 40 mg./ml. in water, but is soluble at less than 1 mg./m1.in methanol and higher alcohols, acetone, chloroform, hexane, anddimethylsulfoxide.

Infrared spectrum: The infrared absorption spectrum of ladakamycinsuspended in mineral oil mull is reproduced in FIG. 1 of the drawing.Ladakamycin shows peaks at the following wave lengths expressed inreciprocal centimeters:

3390 s 1235 M 3320 s) 1216 W 3200 s) 1200 (M) 2950 s 611 1155 M 2920 s611 1137 M 2850 s (oil) 1130 (M) 1968 w 1109 s 1680 s 1073 (M) 1648 s1062 s 1543 w) 1037 (M) 1535 W 1006 M 1530 (M) 987 M 1522 (M) 983 (M)1458 s 6i1 943 (W) 1318 (W) 790 (M) 1298 s 750 W Band intensities areindicated as S, M, and W, respectively, and are approximated in terms ofthe backgrounds in the vicinity of the band. An S band is of the sameorder of intensity as the strongest in the spectrum; M bands are betweenone-third and two-thirds as intense as the strongest band, and W bandsare less than one-third an intense as the strongest band. Theseestimates are made on a basis of a percent transmission scale.

In vitro biological characterization of ladakamycin The inhibition ofEscherichia coli by ladakamycin can be reversed rather specifically byseveral pyrimidine compounds. The most eifective reversing agent wascytidine, as demonstrated by the following experiment. E. coli was grownon petri plates in a completely synthetic medium. Aliquots of suchsynthetic agar were supplemented with different purines and pyrimidinesat levels of 50 mcg./ml. Ladakamycin was applied on 6-mm. paper discs,the plates were incubated at 30 C. for 18 hours, and the resulting zonesof inhibition were measured. The results are as follows:

10 Inhibition by ladakamycin of E. coli grown on various media Zone ofinhibition Medium: in mm. Control-MSM 31 MSM+cytidine 0MSM-i-deoxycytidine 29 MSM+uridine 17 MSM+deoxyuridine 29MSM-i-thymidine 30 MSM+orotic acid 30 MSM+adenosine 28MSM-i-deoxyadenosine 30 MSM+guan0sine 32 MSM+deoxyguanosine 31MSM-l-inosine 31 MSM-I-xanthosine 31 MSM=minimal synthetic medium offollowing composition: K2HPO4, 3.5 g.; I{H2PO4, 1.5 g.; MgSO4'7H2O, 0.1g.; (NH1)2SO4, 1.0 g.; glucose, 2.0 g.; agar, 15.0 g.; distilled water,1 liter. We claim: 1. An antibiotic assaying at least 12 biounits/ml. ofladakamycin, a compound which (a) is effective in inhibiting the growthof various Gram-negative bacteria; and in its essentially purecrystalline form (b) is soluble in water and slightly soluble inmethanol, acetone, chloroform, hexane, and dimethylsulfoxide; (c) hasthe following elemental analyses: C, 39.25;

H, 5.04; N, 22.87; 0, 31.44; (d) has an empirical formula C l-1 N 0 (e)has a characteristic ultraviolet absorption spectrum (f) has a meltingpoint 228230 C.; and

(g) has a characteristic infrared absorption spectrum as shown in FIG. 1of the accompanying drawing.

2. A compound, ladakamycin, according to claim 1, in its essentiallypure form. 3

3. A compound, ladakamycin, according to claim 1, in its essentiallypure crystalline form.

4. A process which comprises cultivating Streptoverticillium ladakanusvar. ladakanus in an aqueous nutrient medium under aerobic conditionsuntil substantial antibacterial activity is imparted to said medium byproduction of ladakamycin.

5. A process which comprises cultivating Streptoverticillium ladakanusvar. ladakanus in an aqueous nutrient medium containing a source ofassimilable carbohydrates and assimilable nitrogen under aerobicconditions until substantial antibacterial activity is imparted to saidmedium by production of ladakamycin and isolating the ladakamycin soproduced.

6. A process according to claim 5 in which the isolation comprisesfiltering the medium and then contacting the filtrate with an adsorbentfor ladakamycin the compound defined in claim 1, and recoveringladakamycin from the adsorbent.

References Cited UNITED STATES PATENTS 3,027,300 3/1962 Bergy et all67--65 JOHNNIE R. BROWN, Primary Examiner US. Cl. X.R.

